Modulation of telomere length in telomerase positive cells and cancer therapy

ABSTRACT

Induction of telomere shortening, G2 arrest and apoptosis in telomerase positive cancer cells using acyclic nucleoside analogs has been disclosed. In addition, methods for impairment or prevention of tumorigenic telomerase positive cells from having a chance to grow into a tumor and methods for promoting tumor regression (decrease in size of an established tumor) using acyclic nucleoside analogs has been disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.11/886,446 filed on Sep. 13, 2007. Priority is claimed based U.S.application Ser. No. 11/886,446 filed on Sep. 13, 2007, which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to the field of cancer therapy.Specifically, the present invention relates to the regulation oftelomere elongation in telomerase positive cells. More particularly, thepresent invention relates to the use of acyclic nucleoside analogsincluding ganciclovir (GCV), acyclovir (ACV) and their ester pro-drugsfor interfering with telomere elongation, for induction of apoptosis andfor treating or preventing telomerase positive cancers.

BACKGROUND OF THE INVENTION

An asymmetry in the synthesis of leading and lagging DNA strands createsthe “end problem” for replication of linear genomes.sup.1 To overcomethis problem, eukaryotic chromosomes have specialized end structures,telomeres, consisting of TTAGGG repeats.sup.2 Telomerase.sup.3,4 is aribonucleoprotein enzyme that elongates telomeres and thereforemaintains chromosomal stability in majority of cancer cells during celldoubling.sup.5 The gradual loss of DNA from the ends of telomeres duringcell doubling has been implicated in the control of cellularproliferative potential in somatic cells.sup.6

Normal cultured human cells have a limited replication potential inculture. Normal cells in culture replicate until they reach a discretepoint at which population growth ceases. This is termed mortality stage1 (M1 stage) and is caused by the shortening of a few telomeres to asize that leads to a growth arrest called cellular senescence. Thisstage can be bypassed by abrogation of the function of p53 and pRB humantumor suppressor genes. The cells then can continue to proliferate withfurther decreases in telomere length until another check point termedmortality stage 2 (M2 stage) or crisis stage. The growth arrest in theM2 stage is caused by balance between the cell proliferation and celldeath rate. At this stage, when most of the telomeres are extremelyshort, end-to-end fusions and chromosomal breakage-fusion cause markedchromosomal abnormalities and apoptosis. Under rare circumstances, acell can escape M2 and become immortal by stabilizing the length of itstelomeres. This occurs through the activation of the enzyme telomeraseor an alternative mechanism of telomere lengthening (ALT).sup.7,8

Human germline.sup.9 and the majority of cancer cells.sup.3 expresstelomerase. Telomerase is a ribonucleoprotein enzyme that elongatestelomeres and, therefore, maintains chromosomal stability in majority ofcancer cells during cell doubling.sup.10 Indeed, elongation of shortenedtelomeres by telomerase is a major mechanism of telomere maintenance inthe human cancer cells. Inhibition of telomerase limits the growth ofhuman telomerase positive cancer cells.sup.11 by decreasing telomerelength.

Elongation of shortened telomeres by telomerase is a well knownmechanism of telomere maintenance in the human cancer cells. From abiological point of view, telomerase elongates telomeres by the additionof repetitive DNA sequences of the TTAGGG-type (telomeric sequences), atthe end of the telomere, during cell division. Through this action,telomerase imparts chromosomal stability and renders the cell immortal.In attempting to obtain selective inhibitors as useful tools forstudying this enzyme, inhibitory effects of nucleotide analogues havebeen investigated in cell-free systems (Yamaguchi et al., (2001, NucleicAcids Research Supplement No. 1 211-212). Since proliferating cellsincluding cancer cells express telomerase activity while normal humansomatic cells do not express telomerase activity at levels sufficient tomaintain telomere length over many cell divisions as seen in cancercells, telomerase is a good target for treating proliferative disordersincluding cancer.

Currently, strategies aimed at selectively treating the cancers fromtelomerase positive cells involve modulation of TERT (Telomerase ReverseTranscriptase) function or length of telomeres by antisense strategy,dominant negative mutants or pharmacological agents (see, Bisoffi etal., Eur J Cancer, 1998, 34: 1242-1249; Roth et al., Leukemia, 2003,17:2410-2417; Damm et al., EMBO J., 2001, 20:6958-6968; U.S. Pat. Nos.6,294,332, 6,194,206, 6,156,763 and 6,046,307). The use of nucleosideanalogs (e.g., AZT) has been attempted to interfere with humantelomerase activity with an aim to treat cancers. The methods disclosedin the prior art administering nucleoside analogs to modify telomeraseactivity, however, are not satisfactory or are not suitable in aclinical setting because their clinical utility is limited by a lowtherapeutic ratio, i.e., the ratio of toxic dose to effective dose.

Prolonged exposure of telomerase positive cell lines to AZT failed toinduce any significant telomere shortening at a concentration of thedrug equal to 100.mu.M (Murakami, J., Nagai. N., Shigemasa. K., Ohama.K. Inhibition of telomerase activity and cell proliferation by a reversetranscriptase inhibitor in gynaecological cancer cell lines. Eur. J.Cancer 35, 1027-1034 (1999) or even 800.mu.M (Gomez D E, Tejera A M,Olivero O A. Irreversible telomere shortening by3′-azido-2′,3′-dideoxythymidine (AZT) treatment. Biochem Biophys ResCommun. 1998; 246(1): 107-10; Tejera A M, Alonso D F, Gomez D E, OliveroO A. Chronic in vitro exposure to 3′-azido-2′,3′-dideoxythymidineinduces senescence and apoptosis and reduces tumorigenicity ofmetastatic mouse mammary tumor cells. Breast Cancer Res Treat. 2001;65(2):93-9).

While peak serum concentration after taking single oral dose of 300 mgof AZT was less than 10.mu.M, and it was rapidly absorbed within 0.5 h(Morse G D, Olson J, Portnore A, Taylor C, Plank C, Reichman R CPharmacokinetics of orally administered zidovudine among patients withhemophilia and asymptomatic human immunodeficiency virus (HIV)infection. Antiviral Res. 1989 March; 11(2):57-65). Standard AZTtreatment is 500 or 600 mg/day in two or three divided doses for adultsaccording to the recommendations of the manufacturer of Retrovir® (AZT).It has been reported that even a short-time exposure to AZT at aconcentration of 5.mu.M induces undesirable toxic effects on mammaliancells in vitro and in vivo (Roskrow M, Wickramasinghe S N. Acute effectsof 3′-azido-3′-deoxythymidine on the cell cycle of HL60 cells. Clin LabHaematol. 1990; 12(2):177-84.). Based on these reports, one can predictthat doses of nucleoside analogs such as AZT high enough to provideantitelomerase and antitumor efficacies can be highly toxic and causedamage to important tissues in humans. Thus, there is need for theidentification of therapeutic nucleoside analogs, which have modulationor inhibitory activity against human telomerase, and development ofmethods of treatment of cancers in which telomerase contributes to theimmortality and undesirable proliferation.

SUMMARY OF THE INVENTION

The present invention provides compositions containing antiviralnucleoside analogs and methods for their use in the modulation,suppression or inhibition of eukaryotic telomerase activity andtreatment of proliferative disorders including cancer. Moreparticularly, the present invention discloses that acyclic nucleosideanalogs or those nucleoside analogs that are active as anti-herpesvirusand anti-cytomegolovirus agents can modify telomerase activity inproliferating cells including cancer cells and thus function asantineoplastic agents. In an aspect of the invention, it has been foundthat treatment of telomerase positive cells with ganciclovir oracyclovir induces progressive telomere loss, G2 phase arrest,chromosomal abnormalities and eventual cell death.

Further, these antineoplastic nucleoside analogs have a surprisingeffect on telomerase in that clinically acceptable levels are sufficientto control telomerase activity and induce cell death in proliferatingcells. These findings now offer new avenues of therapy for treatment orprevention of cancers characterized by telomerase activity (telomerasepositive cancers).

Currently, there are no therapeutic compositions in use that are basedon nucleoside analogs that are acyclic, antitelomerase andantineoplastic. Applicant is the first to provide a disclosureindicating that inhibition of telomerase in vivo using acyclicnucleoside analogs (also referred to herein as inhibitors or antagonistsof telomerase) is therapeutically beneficial. Further, prior to thisdisclosure, there was no consensus by those in the art that one couldpredict that such manipulations would have therapeutic utility.

As telomeres are involved in controlling the cell cycle, cellreplication and aging, nucleoside analog containing compositions of thepresent invention can prevent or control uncontrolled cell growth andthe immortality of tumor cells. The compositions of the presentinvention find particular utility in the treatment of cell proliferativedisorders, and in particular human tumors characterized as havingtelomeres maintained by telomerase.

Thus, in an aspect, the present invention features a method fortreatment of a condition associated with telomerase, particularlyelevated level of telomerase activity in a cell. The method involvesadministering to that cell or a mammal in need of the treatment acomposition containing a therapeutically effective amount of at leastone nucleoside analog that is an acyclic, antitelomerase andantineoplastic agent. The level of telomerase activity can be measuredas described below, or by any other existing method or equivalentmethod. By “elevated level” of telomerase activity, it is meant that theabsolute level of telomerase activity in the particular cell is elevatedcompared to normal cells in that subject or individual, or compared tonormal cells in other subjects or individuals not suffering from thecondition. Examples of such conditions include cancerous conditions, orconditions associated with the presence of cells which are not normallypresent in that individual. In one embodiment, the compositions containnucleoside analogs other than AZT, ddI, ddA, d4T (Strahl C, Blackburn EH Effects of reverse transcriptase inhibitors on telomere length andtelomerase activity in two immortalized human cell lines. Mol Cell Biol.1996; 16(1):53-65). Preferably, the compositions contain GCV or ACV ortheir prodrugs. In another embodiment, these compositions may contain,in addition, clinically acceptable levels of AZT. The utilization ofthese telomerase inhibitors (which either directly inhibit thetelomerase activity or indirectly incorporate into telomere and thusprevent telomere's further elongation) will lead to progressive telomereshortening in tumors where telomerase is active. Once the telomerelength shortens to a critical length, the tumor will go into crisis andeventually die. These telomerase inhibitors should have little or noeffect on the normal somatic cells because telomerase activity in normalcells is generally low or undetectable.

Interference with telomerase activity may either directly result in celldeath or may potentiate the effects of chemotherapeutic agents thatultimately kill cells through apoptosis. In particular, the inventionprovides a method for inhibiting proliferation of telomerase expressingcells having potential for continuous increase in cell number byadministering the inhibitors and antagonists of telomerase.Administration of a nucleoside analog can be achieved by any desiredmeans well known to those of ordinary skill in the art.

In an embodiment of the invention, a method for prevention of a cancercharacterized by expression of telomerase in a mammal or a subject (e.g.a human) in need thereof is provided. The preventive method involvesadministration of a therapeutically effective amount of a composition tothe mammal. The composition has a telomerase inhibitor or antagonist ofthe present invention. The inhibitor or antagonist blocks thelengthening of telomeres in telomerase-positive cells, therebyinhibiting proliferation of telomerase expressing cells. The inhibitoris an acyclic nucleoside analog or a pharmaceutically acceptable salt ofsuch an analog or a liquid or solid food material that is enriched withthe inhibitor or antagonist. The food product can be, for example, afunctional food in the form of butter, margarine, biscuits, bread, cake,candy, confectionery, yogurt or another fermented milk product, orcereal suitable for consumption by humans. Alternatively, it can be anutritional supplement, a nutrient, a pharmaceutical, food, anutraceutical, a health food and/or a designer food. Periodically, thehuman is tested for the presence of telomerase positive cells. The useof inhibitor or antagonist may be stopped once the telomerase positivecells are no longer detected in the mammal.

In addition to the therapeutic aspect, the present invention alsoprovides diagnostic methods and kits for detecting pathologicallyproliferating cells expressing telomerase. These and other embodimentsof the invention will be described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates flow cytometry data showing decrease in telomerelength, massive apoptosis and changes in cell cycle after 10 days oftreatment of telomerase positive HeLa cell line with 1.5.mu.M of GCV.Untreated cells—top panel, treated cells—bottom panel.

FIG. 2 illustrates flow cytometry data showing decrease in telomerelength, massive apoptosis and changes in cell cycle after 14 days oftreatment of telomerase positive NuTu-19 cell line with 3.mu.M of ACV.Untreated cells—top panel, treated cells—bottom panel.

FIG. 3 illustrates flow cytometry data showing changes in the cell cycledistribution in HeLa (top panel) and NuTu-19 (bottom panel) cellstreated with 1.5.mu.M of GCV for 10 days. Untreated cells—grey, treatedcells—dark.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods involving theuse of nucleoside analogs capable of interfering with mammaliantelomerase activity. In particular, it has been found that certainnucleoside analogs can affect telomere/telomerase function in cells atclinically acceptable levels. Specifically, in the context of thisinvention, the “nucleoside analogs” are compounds with structuralsimilarities to the naturally occurring nucleosides but are limited tothose analogs that are acyclic. The acyclic nucleoside analogscontemplated in the present invention are those having a purine (or apyrimidine) skeleton with a tail portion (e.g.,9-(1,3-dihydroxy-2-propoxymethyl present in guanine) but lacking thehydroxyl cyclic ring (pentose). Examples of the analogs of the presentinvention include but are not limited to the following: acyclovir,ganciclovir, penciclovir and the corresponding pro-drugs, i.e.,valacyclovir, valganciclovir and famciclovir, respectively.Acyclovir.sup.12 acts by mimicking a cellular DNA constituent, guanine.That is the “G” in the AT-CG of DNA. Acyclovir(9-[2(hydromethoxy)-methyl] guanine), although structurally similar to“G,” is missing its tail—a hydroxyl “cyclic” ring (pentose) and thus itis “acyclic.” Ganciclovir.sup.13,14,15 and penciclovir.sup.16,17 arealso “acyclic” because they lack the hydroxyl cyclic ring. In anembodiment of the invention, the tail portion of the acyclic nucleosideanalogs of the present invention has at least one hydroxyl groupmimicking the 3′- and 5′-hydroxyl groups of the 2′-deoxyribose moiety ofnucleosides. The acyclic nucleoside analogs of the present inventionhave been found to exhibit antitelomerase and antineoplastic propertieswith clinically acceptable degree of toxicity. The acyclic nucleosideanalogs acyclovir, ganciclovir, penciclovir and the correspondingpro-drugs, i.e., valacyclovir, valganciclovir and famciclovir, are allapproved for clinical use as antiviral drugs. Their chemical structuresand dosage regimens for combating viral infections are well known to oneskilled in the art.

While acyclovir, ganciclovir, penciclovir and the correspondingpro-drugs are well known as antiviral medicines for the treatment ofHerpes virus or/and CMV infections, their use in therapy of neoplasticdiseases is unknown. It is also known in the art that the target enzymefor these anti-herpes virus agents is the DNA polymerase.

In the present invention it has been shown that the acyclic antiviralagents can also target eukaryotic telomerase in proliferating cells andtumors. It is believed that these agents, once inside a proliferatingcell, get phosphorylated (e.g., di- and triphosphate) forms and competewith the natural substrates (e.g., dGTP) of the telomerase reaction. Thephosphorylated analogs can inhibit the incorporation of the naturalsubstrates into the growing telomere DNA chain or can themselves becomeincorporated into DNA thereby interfering with telomerase mediatedpolymerization activity, which eventually leads to termination of chainelongation. In essence, these nucleoside analogs, by termination ofchain elongation, damage telomeric DNA, shorten telomeres and causeapoptosis.

Damage to telomeres is more detrimental to rapidly growing (e.g., tumor)cells than to normal cells.

The anti-HIV and anti-herpes nucleoside analogs have been reported to beactive only after their phosphorylation from the nucleoside to thenucleotide stage. Thus, phosphorylation appears to be a crucial factorfor the activity of nucleoside analogs against their targets. In thisregard, AZT has been reported to require three consecutivephosphorylations for it to be active against telomerase.

The acyclic nucleoside analogs of the present invention are more potentand selective antitelomerase agents than the prior art knownantitelomerase nucleoside analogs such as AZT; clinically acceptabledoses.sup.18,19,20,21,22 are sufficient for realizing antitelomeraseactivity and apoptosis or cell death as compared to the nucleosideanalogs such as AZT.

Induction of telomere shortening, G.sub.2/M arrest (also referred toherein as G.sub.2 arrest) and apoptosis in telomerase positive cancercells after ganciclovir (GCV) and acyclovir (ACV) treatments has beencarried out as described below.

To detect telomerase specific activity in two cell lines (Hela andNuTu-19) real time TRAP assay was performed. The reportedtelomerase-positive cell lines (HeLa) was used for comparison.sup.4 Bothcell lines were positive in this test (data not shown).

The telomerase positive cell lines were treated with therapeuticconcentrations of GCV (1.5.mu.M) or ACV (3.0.mu.M), to demonstrate thattelomeric DNA synthesis could be inhibited within the cells, and therebyinduce telomere shortening. Telomere length in GCV and ACV treated anduntreated cell lines was measured by flow cytometry with atelomere-specific peptide nucleic acid (PNA) probe.sup.23,24. Todetermine cell cycle distribution, cells were stained with propidiumiodide (PI).sup.23. After 14 days of both kinds of treatment, both celllines demonstrated telomere shortening, massive apoptosis and G2 arrest(FIGS. 1 and 2).

To demonstrate changes in cell cycle distribution HeLa and NuTu-19 cellswere treated with GCV or ACV for 14 days stained with PI, and analyzedby flow cytometry simultaneously. Results show G2 arrest of cell cycle(FIG. 3). It is important to note that changes were rapid and could bedetected after only 14 days of ACV treatment. In contrast, thenucleoside analog, AZT had no effect on telomere length or cell cycledistribution in telomerase positive cells, HeLa and NuTu-19, even atelevated concentrations e.g., 100.mu.M (data not shown).

At the same time, PI staining demonstrated a higher DNA content in GCVor ACV treated cells at later stages of treatment, compared to untreatedcells. A rational explanation of this fact is a short telomere inducedchromosome end-to-end joining.sup.25.

The origin of the cell lines are uterine cervix (HeLa) and epithelialovarian (NuTu-19). Cells were cultured in D-MEM media supplemented with10% fetal calf serum at 37.degree. C. in a humidified atmosphere of 5%CO.sub.2. For treatment of the cells with GCV, the media wassupplemented with 1.5.mu.M of GCV (Cymevene, Hoffman-La Roche). Fortreatment of the cells with ACV, the media was supplemented with 3.mu.Mof Aciclovir (Aciclovir, TEVA Pharm. Ind. Ltd, Israel).

Real time TRAP assay was performed as described (Wege et al., SYBR Greenreal-time telomeric repeat amplification protocol for the rapidquantification of telomerase activity. Nucleic Acids Res. 2003;31(2):E3-3).

For telomere length measurement by flow cytometry, cells were stainedwith telomere specific FITC conjugated (C.sub.3TA.sub.2).sub.3 PNA(Applied Biosystems) probe and contrastained with 0.06.mu.g/ml PI asdescribed by Rufer, N., Dragowska, W., Thornbury G., Roosnek, B.,Lansdorp P. M. Telomere length dynamics in human lymphocytesubpopulations were measured by flow cytometry. Nat. Biotechnol. 16,743-747 (1998)).

Thus, it has been demonstrated herein that the nucleoside analogs GCVand ACV clearly block telomerase positive cancer in widely acceptedmodel systems. Useful telomerase-inhibitory compounds are not believedto be limited in any way to the specific compounds or nucleotide analogsand derivatives specifically exemplified above. In fact, it may prove tobe the case that the most useful pharmacological compounds designed andsynthesized in light of this disclosure will be second generationderivatives or further-chemically-modified acyclic nucleoside analogs.

Although not suggesting the advantageous uses made possible by thisinvention, the previous administration of GCV for treating CMV(cytomegalovirus) infections in patients with AIDS or otherimmunodeficiencies means that GCV can be readily administered to cancerpatients.

Further, the present use of a number of acyclic nucleoside analogs toHSV and CMV patients, coupled with the ability to use significantlylower doses of these analogs, should speed up regulatory approval forthe use of acyclovir, ganciclovir, penciclovir and the correspondingpro-drugs, i.e., valacyclovir, valganciclovir and famciclovir, in thetreatment of telomerase induced and/or mediated cancers.

The present invention also encompasses the use of various animal models.By developing or isolating cell lines that express telomerase one cangenerate disease models in various laboratory animals. These models mayemploy the subcutaneous, orthotopic or systemic administration of cellsto mimic various disease states. For example, the HeLa cell line can beinjected subcutaneously into nude mice to obtain telomerase positivetumors. The resulting tumors should show telomerase activity intelomeric repeat amplification protocol (TRAP) assay. Such animal modelsprovide a useful vehicle for testing the nucleoside analogs individuallyand in combinations as well.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria including, but are not limited to,survival, tumor regression, arrest or slowing of tumor progression,elimination of tumors and inhibition or prevention of metastasis.

Treatment of animals with a test compound would involve theadministration of the compound or composition in an appropriate form tothe animal. The pharmaceutical compositions, inhibitory or antagonisticagents of the present invention can be administered in a variety of waysincluding but not limited to oral, parenteral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumoral injection.

The compositions of the present invention would be important in a numberof aspects. They would be important in regimens for the treatment oftelomerase-related cancers, whether administered alone or in combinationwith chemo- and/or radiotherapeutic regimens known to one skilled in theart in the treatment of cancer. Alternatively, by simply reducingtelomerase activity, these compositions will be instrumental inselectively inducing massive apoptosis of cancer cells.

The nucleoside analogs may be administered in a physiologically orpharmaceutically acceptable carrier to a host for treatment ofproliferative diseases, etc. Pharmaceutically acceptable carriers aredetermined in part by the particular composition being administered aswell as by the particular method used to administer the composition.

In an aspect of the present invention, methods for preventing ortreating disorders caused by the presence of inappropriately orpathologically proliferating cells or immortal cells in mammals areprovided. The inappropriately or pathologically proliferating cells orimmortal cells exist and reproduce independently of cells' normalregulatory mechanisms. These cells are pathologic because they deviatefrom normal cells as a result of activity of a cellular element, i.e.,telomerase. Of course, the term “inappropriately proliferating cells” asused herein may be benign hyperproliferating cells but unless statedotherwise these cells refer to malignant hyperproliferating cellscharacteristic of a wide variety of tumors and cancers.

In particular, methods for preventing or treating human tumorscharacterized as expressing telomerase are provided. The human tumorsinclude stomach cancers, osteosarcoma, lung cancers, pancreatic cancers,adrenocortical carcinoma or melanoma, adipose cancers, breast cancers,ovarian cancers, cervical cancers, skin cancers, connective tissuecancers, uterine cancers, anogenital cancers, central nervous systemcancers, retinal cancer, blood and lymphoid cancers, kidney cancers,bladder cancers, colon cancers and prostate cancers. The prevention ortreatment of the disorders, according to the present invention, isachieved by the utilization of acyclic nucleoside analogs (inhibitors orantagonists of telomerase) of the present invention. The inhibitor(s) orantagonist(s) used in the present invention are those that directly orindirectly interact with telomerase to inhibit its activity and/or thosethat get incorporated into telomere and thus prevent telomere fromfurther elongation despite the functional telomerase thereby inhibitingthe growth of cells expressing telomerase. Thus, the inhibitors orantagonists of telomerase are used for inhibiting the growth of cells.For example, when the inhibitors or antagonists of telomerase areadministered to a patient, these cause progressive telomere shortening,cell cycle arrest in the cells and/or massive apoptosis of the cellsexpressing telomerase. In the present invention, the terms “inhibitingthe growth” or “inhibition of growth” may also mean reducing orpreventing cell division. Inhibition of growth of cells expressingtelomerase, in the present invention, may be about 100% or less but not0%. For example, the inhibition may be from about 10% to about 100%,preferably at least about 25%, and more preferably at least about 50%,still more preferably at least about 90%, 95% or exactly 100% comparedto that of the control cells (control cells express telomerase but arenot treated with an inhibitor or antagonist). The inhibition of growthcan be measured by any methods known in the art. For example, viablecell number in treated samples can be compared with viable cell numberin control samples, determined after incubation with vital stains. Inaddition, growth inhibition can be measured by assays that can detectreductions in cell proliferation in vitro or in vivo, such as tritiatedhydrogen incorporation assays, BdU incorporation assay, MTT assay,changes in ability to form foci, anchorage dependence or losingimmortalization, losing tumor specific markers, and/or inability to formor suppress tumors when injected into animal hosts (Dorafshar et al.,2003, J Surg Res., 114:179-186; Yang et al., 2004, Acta Pharmacol Sin.,25:68-75).

The development of a cancerous tumor from a single immortalized cell orfew such cells may take several months to years in humans. By practisingthe present invention, however, cancer can be prevented because thetumorigenic telomearse positive cells treated with telomerase inhibitorslose their proliferative potential before they have had a chance to growinto a tumor. Further, periodic preventative administration oftelomerase inhibitors or antagonists to at risk groups in order to stoptumor progression before clinical manifestation of cancer couldpotentially decrease the rate of new cancer cases significantly.

The nucleoside compounds may be administered either singly or incombinations of different analogs and by any routes of administration,including oral administration. The nucleoside analogs ACV, GCV or theirL-valil esters valganciclovir (V-GCV) and valacyclovir (V-ACV) are thepreferred nucleoside analogs. All of them are commercially available andthe formulations are described in a number of patents and publications.

The cells with telomerase activity should be selectively targetedbecause these cells depend on telomerase for elongating or maintainingtelomeres and the elongation or maintenance of telomeres requires theinteraction of the nuclosides and/or their analogs with telomerase. Tothe extent any specific targeting agent is desired for delivering theanalogs to exert anti-cancer effects, the use of targeted ACV or GCVand/or other analogs are contemplated herein. Accordingly, in someembodiments, pharmaceutical compositions may have the active compound,in this case, ACV and GCV or a other nucleoside analog, which has beenconjugated to a targeting agent (e.g., a peptide) for specific deliveryto particular target cells or to nuclear portion within cells.

The dose of a given inhibitor or antagonist of telomerase can bedetermined by one of ordinary skill in the art upon conducting routineexperiments. Prior to administration to patients, the efficacy may beshown in standard experimental animal models. In this regard any animalmodel for telomerase induced cancer known in the art can be used (Hahnet al., 1999, Nature Medicine, 5(10):1164-1170; Yeager et al., 1999,Cancer Research, 59(17):4175-4179). The subject, or patient, to betreated using the methods of the invention is preferably human, and canbe a fetus, child, or adult. Other mammals that may be treated can bemice, rats, rabbits, monkeys and pigs.

The inhibitors or antagonists can be used alone or in combination withother chemotherapeutics (i.e., non-nucleoside analog based anti-canceragents) including irradiation. For example, therapy of telomeraseinduced cancers may be combined with chemo and/or radiotherapy to treatcancers induced by telomerase or some other factors. Examples ofchemotherapeutic agents known to one skilled in the art include, but arenot limited to, anticancer drugs such as bleomycin, mitomycin, nitrogenmustard, chlorambucil, 5-fluorouracil (5-FU), floxuridine (5-FUdR),methotrexate (MIX), colchicine and diethylstilbestrol (DES). To practicecombined therapy, one would simply administer to an animal an inhibitorcomponent of the present invention in combination with anotheranti-cancer agent (chemo or radiation) in a manner effective to resultin their combined anti-cancer actions within the animal or patient. Theagents would therefore be provided in amounts effective and for periodsof time effective to result in their combined presence in the region oftarget cells. To achieve this goal, the agents may be administeredsimultaneously, and in the case of chemotherapeutic agents, either in asingle composition or as two distinct compositions using differentadministration routes. Alternatively, the two treatments may precede, orfollow, each other by, e.g., intervals ranging from minutes to hours ordays. By way of example, and not limitation, the average daily doses ofGCV for systemic use may be 100 mg/kg per day for human adults, 50 mg/kgper day for mice and human infants.

Some variation in dosage may occur depending on the condition of thesubject being treated. The physician responsible for administration willbe able to determine the appropriate dose for the individual patient andmay depend on multiple factors, such as, the age, condition, filehistory, etc., of the patient in question.

Accordingly, the methods of the invention can be used in therapeuticapplications for conditions and diseases associated with telomeraseinduced pathological proliferation of cells. Diseases that would benefitfrom the therapeutic applications of this invention include all diseasescharacterized by cell hyperproliferation including, for example, solidtumors and leukemias, and non-cancer conditions. It is furthercontemplated that the method of the invention can be used to inhibit thegrowth of cancer cells not only in an in vivo context but also in an exvivo situation. The method of the invention is particularly useful forinhibiting the growth of pathologically proliferating human cells exvivo, including, but not limited to, human cancer cells—osteosarcoma,breast carcinoma, ovarian carcinoma, lung carcinoma, adrenocorticalcarcinoma or melanoma. Bone marrow purging, which is well known incancer therapy area, is an example of ex vivo treatment for inhibitingthe growth of pathologically proliferating human cells.

The present invention provides methods and kits for identifyinginappropriately, pathologically or abnormally proliferating cells due tothe expression of telomerase in the cells. The methods can be used as ascreening method that aids in diagnosing the presence of a cancerouscell or tumor in a patient by determining the presence (and/or level) ofexpression of telomerase in tissue from the patient, the presence oftelomerase expression being indicative of cancer cells or pathologicalcell proliferation in the patient.

For example, cancerous tumor samples can be diagnosed by their inabilityto proliferate in the presence of the acylic nucleoside analogs of thepresent invention. The diagnosis may further involve the detection oftelomerase specific mRNA expression measured by a variety of methodsincluding, but not limited to, hybridization using nucleic acid,Northern blotting, in situ hybridization, RNA microarrays, RNAprotection assay, RT-PCR, real time RT-PCR, or the presence oftelomerase catalytic subunit encoded protein measured by variety ofmethods including, but not limited to, Western blotting,immunoprecipitation or immunohistochemistry, or enzymatic activity oftelomerase (TRAP assay and its modifications.sup.4,26,27).

In a preferred embodiment, nucleic acid probes directed againsttelomerase catalytic subunit RNA can be used to detect presence and/orincreases in telomerase catalytic subunit RNA mRNA levels in tissuesundergoing rapid proliferation, such as primary cancer cells, includinghuman osteosarcoma, breast carcinoma, ovarian carcinoma, lung carcinoma,adrenocortical carcinoma or melanoma. Thus, the present inventionprovides methods of using nucleic acid probes that are complementary toa subsequence of an telomerase to detect and identify pathologicallyproliferating cells, including cancer cells. For example, the method foridentifying a pathologically proliferating cell may involve using anucleic acid probe directed against an hTERT mRNA to compare the levelof expression of hTERT mRNA in a test cell with the level of expressionof hTERT mRNA in a control cell. A test cell is identified as apathologically proliferating cell when the level of hTERT expression isobserved as in the control cell. The nucleic acid probe used in themethod of the invention, however, may also be substantiallycomplementary to an hTERT mRNA sequence of human mouse or other mammal.

It will be apparent to one of ordinary skill in the art thatsubstitutions may be made in the nucleic acid probe which will notaffect the ability of the probe to effectively detect the hTERT mRNA inpathologically proliferating cells (e.g., cancer cells) and thus, suchsubstitutions are within the scope of the present invention. The nucleicacid probe used in the method of the present invention can be a DNAprobe, or a modified probe such a peptide nucleic acid probe, aphosphorothioate probe, or a 2′-O methyl probe. The length of thenucleic acid probe may be from about 8 or 10 to 50 nucleotides,preferably from about 15 to 25 nucleotides in length. The method of theinvention can be readily performed in a cell extract, cultured cell, ortissue sample from a human, a mammal, or other vertebrate.

The methods of the present invention are useful for detecting theinappropriately, pathologically or abnormally proliferating cells due tothe expression of telomerase in the cells in vitro, in cell cultures,and in human cells and tissues, such as solid tumors and cancers (e.g.,human osteosarcoma, breast carcinoma, ovarian carcinoma, lung carcinoma,adrenocortical carcinoma or melanoma).

The present invention also provides kits for detecting and/or inhibitinghyperproliferating cells or cancer cells. The kit can have ACV, GCV,valganciclovir valaciclovir or other acyclic nucleoside analogs and/orhave a nucleic acid probe that is fully or substantially complementaryto a subsequence of an hTERT mRNA.

The pharmaceutical compositions, inhibitory or antagonistic agents ofthe present invention can be administered in a variety of ways includingorally, topically, parenterally e.g. subcutaneously, intraperitoneally,by viral infection, intravascularly, etc. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways.Formulations suitable for oral administration can be liquid solutions.Formulations suitable for parenteral administration (e.g., byintraarticular, intraventricular, intranasal, intravenous,intramuscular, intradermal, intraperitoneal, and subcutaneous routes)include aqueous and non-aqueous, isotonic sterile injection solutions.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, parenterally orintraperitoneally. Oral and parenteral administrations are the preferredmethods of administration. Techniques for formulation and administrationare routine in the art and further details may be found, for example, inRemington's Pharmaceutical Sciences (2000), Gennaro AR(ed), 20thedition, Maack Publishing Company, Easton, Pa.

Therapeutically effective amount (or effective amount) orpharmacologically effective amount are well recognized phrases in theart and refer to that amount of an agent effective to produce theintended pharmacological result. For example, a therapeuticallyeffective amount is an amount sufficient to effect a beneficialtherapeutic response in the patient over time (i.e., to treat a diseaseor condition or ameliorate the symptoms of the disease being treated inthe patient). Therapeutically effective amount of acyclic nucleosideanalog(s) (or a composition thereof) is that amount effective toreproducibly induce telomere shortening, G.sub.2 arrest and/or massiveapoptosis in cancer cells in an assay in comparison to levels inuntreated cells. Therapeutically effective amount of acyclic nucleosideanalog(s) (or a composition thereof) also means an amount of acyclicnucleoside analog(s) that will decrease, reduce, inhibit or otherwiseabrogate the growth of cancer cells. The amount will preferably be anoptimized amount such that the desired effect is achieved withoutsignificant side effects. As described further in detail below, the dosemay also be determined by the efficacy of the particular inhibitor orantagonistic agent employed and the condition of the patient, as well asthe body weight or surface area of the patient to be treated. The sizeof the dose also will be determined by the existence, nature, and extentof any adverse side-effects that accompany the administration of, forexample, a particular agent, vector or transduced cell type to aparticular patient.

Therapeutically effective doses of agent(s) capable of preventing,inhibiting or reducing the incidence of telomerase mediated cancer arereadily determinable using data from cell culture assays disclosedherein and/or from in vivo assays using an animal model. The animalmodel can also be used to estimate appropriate dosage ranges and routesof administration in humans. Experimental animals bearing solid tumorsof human origin (or art-accepted animal models) are frequently used tooptimize appropriate therapeutic doses prior to translating to aclinical environment. Such models are known to be very reliable inpredicting effective anti-cancer strategies. For example, mice bearingsolid tumors are art-accepted mouse models and are widely used inpre-clinical testing to determine working ranges of therapeutic agentsthat give beneficial anti-tumor effects with minimal toxicity. Due tothe safety already demonstrated in art-accepted models, at least withrespect to nucleoside analogs used in the context of telomerase-mediatedcancer, pre-clinical testing of the present invention will be more of amatter of routine experimentation. In vivo efficacy may be predictedusing assays that measure inhibition of tumor formation (progression),tumor regression or metastasis, and the like.

Exemplary in vivo assays of anti-tumor efficacy of ACV and/or GCV usingnude mice subcutaneous (s.c.) tumors grown from the human HeLa cancercell line (i.e., xenografts bearing mice) as cancer models are describedbelow.

Human cancerous cells needed for in vivo assays may be prepared, forexample, as follows: Telomerase positive HeLa human cell line can beobtained from public sources. Cells are maintained in D-MEM mediasupplemented with 10% foetal calf serum at 37.degree. C. in a humidifiedatmosphere of 5% CO.sub.2.

For in vivo assay, appropriate host, e.g., nude (nu/nu) mice of about5-7 weeks old are obtained and maintained in pathogen-free conditions.Approximately, 1.times.10.sup.6 HeLa cells contained in 200.mu.1 ofserum-free media are delivered to all animals, briefly anaesthetizedwith Metofane, by subcutaneous (s.c.) injection in flank. Then the miceare divided into experimental group and control group.

In one embodiment, impairment of s.c. tumor growth or time toprogression rather than decrease in size of an established tumor isassessed. In this embodiment, starting from the day zero, mice in theexperimental group receive GCV in drinking water ad libitum.Concentration of GCV in water can be 2 mg/ml. Fresh solution of GCV issupplied every 3 days. Mice in the control group receive only drinkingwater. Tumors are measured every 2-3 days. Mice are sacrificed whentumors exceed 1 cm.sup.3. Tumor volume is calculated with formula4/3.pi.r.sup.3, where r is the radius of the tumor. All mice in thecontrol group should develop tumors and all mice in the experimentalgroup remain tumor free.

An in vivo was carried out as follows: Nude mice were injected s.c. withHeLa cells (3.times.10.sup.5) to demonstrate prevention of developmentand treatment of telomerase positive tumors in vivo. Human cancer HeLacell culture was purchased from ATCC. In all, 12 CD1/-nu and 12NMRiI/-nu nude mice were purchased from Charles River Laboratories,Charles River Deutschland GmbH. These nude mice were injected s.c. with3.times.105 HeLa cells. Experimental group received valganciclovir indrinking water from day 0. Specifically, mice in the experimental groups(6 mice per strain) were exposed to Valcyte (val-ganciclovir) indrinking water (1 mg/ml) from day 0. All mice in control and treatedgroups had developed tumors. In about 14 days, all mice were bearing thetumors. The tumor in one mouse from the treated group began to regressand, by about the 30.sup.th day, this tumor was eliminated bymonotherapy with Valcyte. Other mice in the treated groups demonstratedslowing of tumor growth (stabilization).

In another embodiment, the reagents and methods of the invention can beused to promote tumor regression in vivo in immunocompetent animalscarrying pre-established tumors; i.e., the reagents of the invention canbe used to treat animals with pre-existing tumors. In this case, thecancerous 10.sup.6 NuTu-19 cells are injected subcutaneously in theflank of the Fischer rats to establish tumors. Once tumors areestablished after tumor cell implantation, the rats in the experimentalgroup are administered with a composition containing GCV (or ACV) i.g.solution in drinking water ad libitum, and the rats in the control groupreceive the same composition but without the drug (e.g., distilled waterTumor growth is monitored every 2-3 days. When GCV (or ACV) isadministered 21-28 days to these tumor bearing animals, retarded tumorgrowth is observed. Such inhibition of tumor cell growth is not observedin the control group. Few weeks after the start of the treatment, onlythe animals treated with GCV show 100% survival.

In another embodiment, in vivo assays that qualify the promotion ofapoptosis may also be used. In this embodiment, xenograft bearinganimals treated with the therapeutic composition may be examined for thepresence of apoptotic foci and compared to untreated controlxenograft-bearing animals. The extent to which apoptotic foci are foundin the tumors of the treated animals provides an indication of thetherapeutic efficacy of the composition.

From the above exemplary in vivo assays, it should be apparent that thecombination treatment that uses non-nucleoside analog based anti-canceragents (such as, for example, bleomycin, mitomycin, nitrogen mustard,chlorambucil, 5-fluorouracil (5-FU), etc., listed above) or irradiationis not a requirement of the invention. The use of acyclic nucleosideanalog(s) alone or in combination with nucleoside analogs that are notacyclic is sufficient to induce telomere shortening, G.sub.2 arrestand/or massive apoptosis in cancer cells and thus is sufficient toachieve more than a mere weak growth delay of tumors (or sufficient todramatically reduce the growth delay of tumors). Accordingly, in someaspects, the present invention does not involve the use ofnon-nucleoside analog based anti-cancer agents or irradiation. Also, insome aspects of the present invention (methods of treatment orprevention of tumor growth), the following nucleoside analogs areexcluded or not used: HPMPC [(S)-1-[3-hydroxy-2-(phosphomethoxy)propyl]cytosine]; HPMPA which is an adenine derivative or9-(2-[phosphonylmethoxyethyl) (PMEA, adefovir), which are derivatives ofadenine, or guanine (PMEG), 2-6 diaminopurine (PMEDAP), cyclo-propylPMEDAP (cPr-PMEDAP).

This invention encompasses the use of telomerase inhibitors-based cancertherapy for a wide variety of tumors and cancers affecting skin,connective tissues, adipose, breast, lung small cell lung carcinomas andnon-small cell lung cancer (NSCLC)), stomach (gastric cancer), pancreas,ovary, cervix, uterus, kidney, bladder, colon, prostate, anogenital,central nervous system (CNS), retina and blood and lymph (lymphomasresulting from the expression of CDK9/CYCLIN T1 in precursor T cells,precursor B cells, germinal center cells, activated T cells orReed-Sternberg cells), virus-associated cancers (HBV-associated cancers,EBV-associated cancers HCV-associated cancers and HPV-associatedcancers) known in the art, and other cancers mentioned elsewhere in thisdisclosure. In an aspect, however, the present invention does notinclude treatment or prevention of virus-associated cancers known in theart.

In designing appropriate doses of agent(s) for the treatment of humantelomerase-mediated caners (both early stage tumors and vascularizedtumors), one may readily extrapolate from the animal studies describedherein in order to arrive at appropriate doses for clinicaladministration. To achieve this conversion, one would account for themass of the agents administered per unit mass of the experimental animaland, preferably, account for the differences in the body surface areabetween the experimental animal and the human patient. All suchcalculations are well known and routine to those of ordinary skill inthe art. Thus, the determination of a therapeutically effective dose iswell within the capability of those skilled in the art.

For example, in taking the successful doses of GCV or ACV (V-GCV orV-ACV) in cell culture assays and in the mouse studies, and applyingstandard calculations based upon mass and surface area, effective dosesfor use in adult human patients would be between about 1000 mg and about6000 mgs of GCV or ACV per patient per day, and preferably, betweenabout 500 mgs and about 1000 mgs of V-GCV or V-ACV per patient per day.Accordingly, using this information, it is contemplated herein that lowdoses of therapeutic agents (e.g., acyclovir, ganciclovir, penciclovirand the corresponding pro-drugs, i.e., valacyclovir, valganciclovir andfamciclovir) for human administration may be about 1, 5, 10, 20, 25 orabout 30 mgs or so per patient per day; and useful high doses oftherapeutic agent for human administration may be about 250, 300, 400,450, 500 or about 600 mgs or so per patient per day. Useful intermediatedoses may be in the range from about 40 to about 200 mgs or so perpatient

Notwithstanding these stated ranges, it will be understood that, giventhe parameters and detailed guidance presented herein, furthervariations in the active or optimal ranges will be encompassed withinthe present invention. The intention of the therapeutic regimens of thepresent invention is generally to produce significant anti-tumor effectswhilst still keeping the dose below the levels associated withunacceptable toxicity. In addition to varying the dose itself, theadministration regimen can also be adapted to optimize the treatmentstrategy. A currently preferred treatment strategy is to administerbetween about 1-500 mgs, and preferably, between about 10-100 mgs of theinhibitor or antagonist of telomerase or therapeutic cocktail containingsuch, about −4 times within about a 60 days period. For example, doseswould be given on about day 1, day 3 or 4 and day 6 or 7. Administrationcan be accomplished via single or divided doses taken orally or, forexample, by administration to the site of a solid tumor directly or in aslow release formulation. The physician responsible for administrationwill, in light of the present disclosure, be able to determine theappropriate dose for the individual subject, the form and route ofadministration. Such optimization and adjustment are routinely carriedout in the art and by no means reflect an undue amount ofexperimentation. In administering the particular doses themselves, onewould preferably provide a pharmaceutically acceptable compositionaccording to regulatory standards of sterility, pyrogenicity, purity andgeneral safety to the human patient systemically. Physical examination,tumor measurements, and laboratory tests should, of course, be performedbefore treatment and at intervals up to one to few months after thetreatment and one skilled in the art would know how to conduct suchroutine procedures. Clinical responses may be defined by any acceptablemeasure. For example, a complete response may be defined by thedisappearance of all measurable tumors within a given period aftertreatment.

The references numbered 1-27 below are cited in the above description(with the corresponding superscript numbers) and as such one skilled inthe art would match the references to the appropriate superscriptnumbers in the text above. [0073] 1. Olovnikov, A. M. Principle ofmarginotomy in template synthesis of polynucleotides. Dokl. Akad. NaukSSSR 201, 1496-1499 (1971). [0074] 2. Allshire, R. C., Dempster, M.,Hastie, N. D. Human telomeres contain at least three types of G-richrepeat distributed non-randomly. Nucleic Acids Res. 17, 4611-4627(1989).

3. Greider, C. W., Blackburn, E. H. Identification of a specifictelomere terminal transferase activity in Tetrahymena extracts. Cell 43,405-413 (1985). [0076] 4. Morin G B. The human telomere terminaltransferase enzyme is a ribonucleoprotein that synthesizes TTAGGGrepeats. Cell. 1989 Nov. 3; 59(3):521-9. [0077] 5. Kim, N. W.,Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D. Specificassociation of human telomerase activity with immortal cells and cancer.Science 266, 2011-2015 (1994). [0078] 6. Harley, C. B., Futcher, A. B.,Greider, C. W. Telomeres shorten during ageing of human fibroblasts.Nature 34, 458-460 (1990). [0079] 7. Bryan, T. M., Englezou, A.,Dalla-Pozza, L., Dunham, M. A., Reddel, R. R. Evidence for analternative mechanism for maintaining telomere length in human tumorsand tumor-derived cell lines. Nat. Med. 3, 1271-1274 (1997). [0080] 8.Reddel, R. R., Bryan, T. M., Colgin, L. M., Perrem, K. T., Yeager, T. R.Alternative lengthening of telomeres in human cells. Radiat. Res. 155,194-200 (2001). [0081] 9 Wright, W. E., Piatyszek, M. A., Rainey, W. E.,Byrd, W., Shay, J. W. Telomerase activity in human germline andembryonic tissues and cells. Dev. Genet. 18, 173-179 (1996). [0082] 10.Greider C W Mammalian telomere dynamics: healing, fragmentationshortening and stabilization. Curr Opin Genet Dev. 1994; 4(2):203-11.[0083] 11. Hahn, W. C. et al. Inhibition of telomerase limits the growthof human cancer cells. Nat. Med. 5, 1164-1170 (1999). [0084] 12. Elion,G. B.; Furman, P. A.; Fyfe, J. A.; de Miranda, P.; Beauchamp, L.;Schaeffer, H. J. Selectivity of Action of an Antiherpetic Agent,9-(2-Hydroxyethoxymethyl)guanine. Proc. Natl. Acad. Sci. U.S.A. 1977,74, 5716-5720. [0085] 13. Martin, J. C.; Dvorak, C. A.; Smee, D. F.;Matthews, T. R.; Julien, P. H.; Verheyden, J. P. H.9-[(1,3-Dihydroxy-2-propyloxy)methyl] guanine: A New Potent andSelective Antiherpes Agent. J. Med. Chem. 1983, 26, 759-761. [0086] 14.Smee, D. P.; Martin, J. C.; Verheyden, J. P. H.; Matthews, T. R.Antiherpesvirus Activity of the Acyclic Nucleosides9-(1,3-Dihydroxy-2-propoxymethyl)guanine. Antimicrob. Agents Chemother.1983, 23, 676-682. [0087] 15. Field, E. K.; Davies, M. E.; DeWitt, C.;Perry, H. C.; Liou, R.; Germershausen, J.; Karkas, J. D.; Ashton, W. T.;Johnston, D. B.; Tolman, R. L.9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: A SelectiveInhibitor of Herpes Group Virus Replication. Proc. Natl. Acad. Sci.U.S.A. 1983, 80, 4139-4143. [0088] 16. Hamden, M. R.; Jarvest, R. L.;Bacon, T. H.; Boyd, M. R. Synthesis and Antiviral Activity of9-[4-Hydroxy-3-(hydroxymethyl)but-1-yl] purines. J. Med. Chem. 1987, 30,1636-1643 [0089] 17. Vere Hodge, R. A.; Perkins, R. M. Mode of Action of9-(4-Hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) against HerpesSimplex Virus in MRC-5 Cells. Antimicrob. Agents Chemother. 1989, 33,223-229 [0090] 18. de Miranda P, Whitley R J, Blum M R, Keeney R E,Barton N, Cocchetto D M, Good S, Hemstreet G P 3rd, Kirk L E, Page D A,Elion G B. Acyclovir kinetics after intravenous infusion. Clin PharmacolTher. 1979; 26(6):718-28. [0091] 19. Van Dyke R B, Connor J D, Wybomy C,Hintz M, Keeney R E. Pharmacokinetics of orally administered acyclovirin patients with herpes progenitalis. Am J Med. 1982; 73(1A):172-5.[0092] 20. Lycke J, Malmestrom C, Stahle L. Acyclovir levels in serumand cerebrospinal fluid after oral administration of valacyclovir.Antimicrob Agents Chemother. 2003; 47(8):2438-41. [0093] 21. Piketty C,Bardin C, Gilquin J, Gairard A, Kazatchkine M D, Chast F. Monitoringplasma levels of ganciclovir in AIDS patients receiving oral gancicloviras maintenance therapy for CMV retinitis. Clin Microbiol Infect. 2000;6(3):117-20. [0094] 22. Brown F, Banken L, Saywell K, Arum I.Pharmacokinetics of valganciclovir and ganciclovir following multipleoral dosages of valganciclovir in HIV- and CMV-seropositive volunteers.Clin Pharmacokinet. 1999; 37(2):167-76. [0095] 23. Rufer, N., Dragowska,W., Thornbury G., Roosnek, E., Lansdorp P. M. Telomere length dynamicsin human lymphocyte subpopulations measured by flow cytometry. Nat.Biotechnol. 16, 743-747 (1998). [0096] 24. Hultdin, M. et al. Telomereanalysis by fluorescence in situ hybridization and flow cytometry.Nucleic Acids Res. 26, 3651-3656 (1998). [0097] 25. Guiducci, C.,Cerone, M. A., Bacchetti, S. Expression of mutant telomerase in immortaltelomerase-negative human cells results in cell cycle deregulation,nuclear and chromosomal abnormalities and rapid loss of viability.Oncogene 20, 714-725 (2001). [0098] 26. TRAP-ELISA A. K. Velin, A.Herder, K. J. Johansson et al., Telomerase is not activated in humanhyperplastic and adenomatous parathyroid tissue. Eur J Endocrinol 145(2001), pp. 161-164. [0099] 27. real time TRAP (Wege et al., SYBR Greenreal-time telomeric repeat amplification protocol for the rapidquantification of telomerase activity. Nucleic Acids Res. 2003;31(2):E3-3).

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

1. A method for treating a telomerase positive cancer in a human, themethod comprising administering a therapeutically effective amount of acomposition comprising acyclovir or a pharmaceutically acceptable saltthereof to the human suffering from the cancer, wherein thetherapeutically effective amount of said composition is that amounteffective to reproducibly induce telomere shortening, G₂ arrest and/ormassive apoptosis in cancer cells in an assay in comparison to levels inuntreated cells, wherein said acyclovir or a pharmaceutically acceptablesalt thereof induces telomere shortening in said cells.
 2. The method ofclaim 1, wherein said acyclovir or a pharmaceutically acceptable saltthereof is administered in combination with a different type ofnucleoside analog selected from the group consisting of:3′-azido-2′,3′-dideoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), and2′,3′-didehydro-3′-deoxythymidine (d4T), wherein the different type ofnucleoside analog is present in a low dose, which alone is insufficientto treat cancer.
 3. The method of claim 2, wherein the cancer isselected from the group consisting of: bone cancer, breast cancer,prostate cancer, liver cancer, pancreatic cancer, lung cancer, braincancer, ovarian cancer, uterine cancer, testicular cancer, skin cancer,leukemia, melanoma, esophageal cancer, stomach cancer, colon cancer,retinal cancer, or bladder dancer.
 4. The method of claim 3, wherein thecancer is breast cancer.
 5. The method of claim 3, wherein the cancer isovarian cancer.
 6. The method of claim 3, wherein the cancer is uterinecancer.
 7. The method of claim 3, wherein the different type ofnucleoside analog is AZT.
 8. The method of claim 3, wherein thedifferent type of nucleoside analog is ddI.
 9. A method for treating atelomerase positive cancer in a human, the method comprisingadministering a therapeutically effective amount of a compositioncomprising penciclovir or a pharmaceutically acceptable salt thereof tothe human suffering from the cancer, wherein the therapeuticallyeffective amount of said composition is that amount effective toreproducibly induce telomere shortening, G₂ arrest and/or massiveapoptosis in cancer cells in an assay in comparison to levels inuntreated cells, wherein said acyclovir or a pharmaceutically acceptablesalt thereof induces telomere shortening in said cells.
 10. The methodof claim 9, wherein said acyclovir or a pharmaceutically acceptable saltthereof is administered in combination with a different type ofnucleoside analog selected from the group consisting of:3′-azido-2′,3′-dideoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), and2′,3′-didehydro-3′-deoxythymidine (d4T), wherein the different type ofanalog is present in a low dose, which alone is insufficient to treatcancer.
 11. The method of claim 10, wherein the cancer is selected fromthe group consisting of: bone cancer, breast cancer, prostate cancer,liver cancer, pancreatic cancer, lung cancer, brain cancer, ovariancancer, uterine cancer, testicular cancer, skin cancer, leukemia,melanoma, esophageal cancer, stomach cancer, colon cancer, retinalcancer, or bladder cancer.
 12. The method of claim 11, wherein thecancer is breast cancer.
 13. The method of claim 11, wherein the canceris ovarian cancer.
 14. The method of claim 11, wherein the cancer isuterine cancer.
 15. The method of claim 11, wherein the different typeof nucleoside analog is AZT.
 16. The method of claim 11, wherein thedifferent type of nucleoside analog is ddI.
 17. A method of identifyinga composition potentially effective against a predetermined cancercondition, comprising: (a) treating ex vivo a cell indicative of saidcancer condition from a human with a composition comprising one or moreacyclic nucleoside analogs under a given assay conditions, wherein saidnucleoside analogs are selected from the group consisting of: acyclovirand penciclovir, or a prodrug thereof; (b) treating ex vivo another cellindicative of said cancer condition from the human with a compositionnot comprising said one or more acyclic nucleoside analogs under thegiven assay conditions; (c) identifying the composition comprising oneor more acyclic nucleoside analogs as potentially effective against thepredetermined cancer condition if the treatment (a) caused telomereshortening, G₂ arrest and/or massive apoptosis in the cell in comparisonto the treatment (b).
 18. The method of claim 17, wherein the cancer isselected from the group consisting of: bone cancer, breast cancer,prostate cancer, liver cancer, pancreatic cancer, lung cancer, braincancer, ovarian cancer, uterine cancer, testicular cancer, skin cancer,leukemia, melanoma, esophageal cancer, stomach cancer, colon cancer,retinal cancer, or bladder cancer.
 19. The method of claim 18, whereinthe cancer is ovarian cancer.
 20. The method of claim 18, wherein thecancer is uterine cancer.